Use of pirna-54265 in diagnosis, treatment, and prognostic evaluation of colorectal cancer

ABSTRACT

The present invention relates to a marker piRNA-54265 and a method in diagnosis, treatment, and prognostic evaluation of colorectal cancer. The marker piRNA-54265 is capable of being used for diagnosing and/or treating colorectal cancer, wherein the marker piRNA-54265 is selected from any of molecules as follows: (1) SEQ ID NO.29: tggaggtgatgaactgtctgagcctgacc; (2) SEQ ID NO.30: UGGAGGUGAUGAACUGUCUGAGCCUGACC; (3) SEQ ID NO.31: GGUCAGGCUCAGACAGUUCAUCACCUCCA; (4) a piR-54265 variant and a piR-54265 derivative modified from the molecule shown in (1), (2) or (3) with a same function; (5) a piR-54265 polynucleotide construct capable of down-regulating an amount of the piR-54265 in vivo after being introduced; and (6) an expression vector containing the polynucleotide construct of (5). The method is used for diagnosing/screening colorectal cancer, evaluating chemosensitivity of a colorectal cancer patient, evaluating a prognosis of a colorectal cancer patient or treating colorectal cancer.

CROSS-REFERENCE TO RELATED APPLICATION

This is a continuation-in-part application of International ApplicationNo. PCT/CN2018/096398, filed on Jul. 20, 2018, which claims the prioritybenefits of China Application No. 201810146715.6, filed on Feb. 12, 2018and Chinese application no. 201910036674.X, filed on Jan. 15, 2019. Theentirety of each of the above-mentioned patent applications is herebyincorporated by reference herein and made a part of this specification.

BACKGROUND Technical Field

The present invention belongs to the field of biomedical technologies,and more particularly, relates to use of piRNA-54265 in diagnosis,treatment, and prognostic evaluation of colorectal cancer.

Description of Related Art

Colorectal cancer is a common gastrointestinal tumor worldwide, rankinghigh among various malignant tumors, and is one of the leading causes ofcancer-related death around the world. It is clinically difficult tocure advanced colorectal cancer but not colorectal cancer at earlystage. Therefore, early detection and diagnosis of colorectal cancer isof great significance for improving the curative effect and decreasingthe morbidity. Colonoscopy is an important means for early diagnosis ofcolorectal cancer and has high accuracy, but it is an examination methodcausing wounds and is difficult to apply in the general populationcensus due to high economic costs, requirements on operators andequipment and poor compliance of persons to do the examination. Inaddition, there are some early screening and companion diagnosticmethods, but their sensitivity and specificity are poor. Therefore, itis of great demand and significance to develop and establish a lesseconomical cost, technically simple, but sensitive and specificbiomarker for early screening and diagnosis of colorectal cancer.

Meanwhile, chemotherapy for colorectal cancer is also plagued by drugresistance in tumor. According to the estimation of American CancerSociety, more than 90% of cancer patients die from some degrees of drugresistance, and the drug resistance in tumor has become the key factorfor whether or not tumor chemotherapy is successful. There aresignificant individual differences in the effects of chemotherapy, butthe mechanism is unclear and basically there are currently no biomarkersfor predicting curative effect. In addition, there is a huge individualdifference in the prognosis of colorectal cancer patients, and there iscurrently no indicator for evaluating prognosis. Therefore, elucidationand development of biomarkers for colorectal cancer curative effect andprognosis assessment are essential for precise treatment. Anotherimportant issue for colorectal cancer is the judgment of prognosis forpatient treatment and the selection of right treatment solutions.

Therefore, the development of early screening, early diagnosis,treatment and prognosis of colorectal cancer, as well as therapeutictargets and related technologies are urgently needed, and have importantsignificance and application prospects.

SUMMARY

The technical problem to be solved by the present invention is toovercome the defects and deficiencies of the existing diagnosis andtreatment techniques for colorectal cancer, and to provide a newcolorectal cancer diagnostic marker piRNA-54265 (PIWI-interactingRNA54265), which can be used for early diagnosis and early screening,and can specifically indicate the chemosensitivity and prognosis of acolorectal cancer patient, which is helpful for individualization andprecision of clinical treatments; and can further be used as atherapeutic target for colorectal cancer and used for preparing atherapeutic drug for colorectal cancer. Further, the present inventionalso constructs a kit for early screening, diagnosis, curative efficacymonitoring and prognostic evaluation of colorectal cancer with themarker piRNA-54265 as a target.

An objective of the present invention is to provide a marker piRNA-54265capable of being used for diagnosis and/or treatment of colorectalcancer.

Another objective of the present invention is to provide use of themarker piRNA-54265 in early screening, diagnosis, curative efficacymonitoring and prognostic evaluation of colorectal cancer.

One another objective of the present invention is to provide piRNA-54265detection primer sets and kit for early screening, diagnosis, curativeefficacy monitoring and/or prognostic evaluation of colorectal cancer.

The above-mentioned objectives of the present invention are achieved bythe following technical solutions.

Upon research findings of the present invention: there are statisticallysignificant differences in expression of the piRNA-54265 in colorectalcancer and para-carcinoma tissues, and the piRNA-54265 is highlyexpressed in cancer tissues. The expression level of the piRNA-54265 inthe cancer tissues is related to the survival prognosis of thecolorectal cancer patients, and the colorectal cancer patients with highlevel of piR-54365 has poorer prognosis. The piRNA-54265 can be detectedin serums of the colorectal cancer patients, and the content of thepiRNA-54265 in the serums is positively correlated with the content ofthe piRNA-54265 in the cancer tissues, and patients with high contenthave short survival time. Besides, the content of the serum piR-54265 innormal people is significantly lower than the colorectal cancer patient,and thus it is easy to distinguish. With piRNA-54265 knockdown, theabilities of proliferation, migration and invasion of colorectal cancercells are significantly weakened, and the growth and metastasis ofxenograft tumors in nude mice transplanted with colorectal cancer cellscan also be significantly inhibited. When perturbing the expressionlevel of the piRNA-54265, the colorectal cancer cells show a certaindegree of enhancement of chemosensitivity; and the patients withhigh-expressed piRNA-54265 has poor short-term efficacy of chemotherapy.Moreover, the serum piR-54265 level can effectively predict the onset ofcolorectal cancer, and the serum piR-54265 can be found elevated for upto 3 years before the diagnosis of cancer. Early screening and earlydiagnosis of colorectal cancer can be conducted in general populationwith serum piR-54265 detection. Therefore, the piRNA-54265 can be usedas a biomarker for early screening, diagnosis, efficacy evaluation andprognosis judgment of colorectal cancer.

Therefore, all the following use shall fall within the scope ofprotection of the present invention:

Use of the piRNA-54265 as a marker for diagnosis of colorectal cancer.The diagnosis includes early diagnosis and early screening.

Use of the piRNA-54265 as a prognostic evaluation marker for colorectalcancer.

Use of the piRNA-54265 as a chemosensitivity evaluation marker of acolorectal cancer patient.

Preferably, the piRNA-54265 refers to piRNA-54265 in serum or plasma,which can be easily detected by a blood specimen.

Use of the piRNA-54265 as a therapeutic target for colorectal cancer.

Use of the piRNA-54265 in making therapeutic agents or drugs forcolorectal cancer.

Use of an inhibitor or a knockout reagent of the piRNA-54265 as atherapeutic drug for colorectal cancer.

Use of the inhibitor or the knockout reagent of the piRNA-54265 as anancillary drug for improving the chemotherapeutic efficacy of colorectalcancer.

Preferably, the above-mentioned drug is a drug capable of inhibitingproliferation, invasion and/or metastasis of colorectal cancer cells.

In addition, when the piR-54265 is used as an inhibitor, an effectivesequence is a reverse complementary sequence of an original sequence.Therefore, any form of modification taking the sequence as a basis or asan active ingredient shall be protected, i.e., a piRNA-54265 capable ofbeing used for diagnosis and/or treatment of colorectal cancer shallalso fall within the scope of protection of the present invention, andis specifically selected from one of molecules (1) to (6) as follows:

(1) SEQ ID NO. 29: tggaggtgatgaactgtctgagcctgacc, (2) SEQ ID NO. 30:UGGAGGUGAUGAACUGUCUGAGCCUGACC, (3) SEQ ID NO. 31:GGUCAGGCUCAGACAGUUCAUCACCUCCA,

(4) a piR-54265 variant and a piR-54265 derivative modified from themolecule shown in (1), (2) or (3) with a same function;

Manner of modification including but not limited to: methylatedmodification, hydrocarbyl modification, galactosylated modification(such as 2-methoxy-glycosyl modification, hydrocarbyl-glycosylmodification, sugar-ring modification, etc.), nucleination modification,peptide modification, lipid modification, halogen modification, nucleicacid modification (such as “TT” modification), or the like;

(5) a piR-54265 polynucleotide construct capable of affectingtranscription of a corresponding genome after being introduced, therebyendogenously regulating the amount of the piR-54265; and

(6) an expression vector containing the polynucleotide construct of (5).

The piRNA-54265 complementary sequence provided according to the presentinvention, can be introduced and processed into a polynucleotideconstruct which can affect the expression of the correspondingpiR-54265, and the polynucleotide construct is capable ofdown-regulating the amount of the corresponding piRNA in vivo. Thepolynucleotide construct containing the piRNA-54265 complementarysequence can also be inserted into a cellular genome so as tocontinuously generate a transcription product that can specificallyinhibit or degrade the piRNA.

Typically, the polynucleotide construct can be located on an expressionvector. Accordingly, the present invention further includes a vectorincluding the piRNA, or the polynucleotide construct. Usually, theexpression vector further contains a promoter, an origin of replication,and/or a marker gene and the like. Methods well known to those skilledin the art can be used to construct the expression vector required forthe present invention. These methods include DNA recombinationtechniques in vitro, DNA synthesis techniques, recombination techniquesin vivo, and the like. The expression vector preferably includes one ormore selectable marker genes to provide phenotypic character forselection of transformed host cells, such as kalamycin, gentamicin,hygromycin, ampicillin resistance, etc. Moreover, the polynucleotideconstruct is not limited to other genome or transcriptome regulatingmethods in the art, such as CRISPR-Cas9.

In addition, based on the above studies, the piR-54265 related to thepresent invention can be used as a specific molecular marker for cancerincidence or risk prediction, early screening, early diagnosis,therapeutic sensitivity, and prognostic evaluation of cancer, and atherapeutic target for cancer.

Therefore, the present invention provides a method fordiagnosing/screening colorectal cancer, including detecting theexpression level of piRNA-54265 in a specimen under test (such astissue, serum, plasma and even other types of specimens like urine andsaliva), and judging whether the specimen to be detected suffers fromthe colorectal cancer or has a risk of suffering from the colorectalcancer according to the expression level of the piRNA-54265.

A method for evaluating chemosensitivity of a colorectal cancer patientincludes detecting the expression level of piRNA-54265 in a specimenunder test (such as tissue, serum, plasma or other types of specimens),and evaluating the chemosensitivity of the patient according to theexpression level of the piRNA-54265.

A method for evaluating prognosis of a colorectal cancer patient ischaracterized by detecting the expression level of piRNA-54265 in aspecimen under test (such as tissue, serum, plasma or other types ofspecimens), and evaluating the prognosis of the patient according to theexpression level of the piRNA-54265.

Methods for detecting the expression level of the piRNA-54265 are notstrictly limited, and all the methods in the art capable of achievingthe detection objects can be used and shall fall within the scope ofprotection of the present invention.

Specifically, the methods for detecting the expression level of thepiRNA-54265 include, but are not limited to molecular detectiontechniques, and all the methods and use involving direct and/orindirect, qualitative and/or quantitative detection of the piR-54265shall fall within the scope of protection of the present invention,including specific primers and/or probes for detecting piR-54265.

The present invention further provides a method for treating colorectalcancer, including performing gene therapy with piRNA-54265 as a targetto inhibit or silence expression of the piRNA-54265, so as to realize apurpose of treating and assisting in treating colorectal cancer.

Methods for performing gene therapy on the colorectal cancer with thepiRNA-54265 as the target are not strictly limited, and all the methodsin the art capable of achieving the gene therapy object can be used andshall fall within the scope of protection of the present invention.

In addition, as an alternative implementation, the present inventionfurther provides a specific solution for detecting piRNA-54265, i.e.,quantitative PCR detection, which provides a specific primer designpattern for piRNA-54265 and constructs a kit. The specific solution isas follows:

a piRNA-54265 detection kit for early screening, diagnosis, curativeefficacy monitoring and prognosis evaluation of colorectal cancerincludes primer set for specifically detecting piRNA-54265, wherein theprimers are a quantitative PCR primer pair which includes a forwardprimer and a reverse primer, and the forward primer is designed based onthe sequence of the piRNA-54265 (which changes U in the RNA sequence ofthe piRNA-54265 to T), and sequentially includes an amount of bases fromthe 5′⁻ of the sequence, and shall include the first five bases “TGGAG”at least.

As a preferred selectable solution, the primer pair can be a piRNA-54265stem-loop PCR primer pair, or a piRNA-54265 PolyA tailed PCR primerpair.

Preferably, the piRNA-54265 stem-loop PCR primer pair is shown as SEQ IDNO.8/9, SEQ ID NO.10/9, SEQ ID NO.11/9, SEQ ID NO.12/13, SEQ IDNO.14/13, SEQ ID NO.15/13, SEQ ID NO.16/13, SEQ ID NO.17/13, SEQ IDNO.18/13, SEQ ID NO.19/13 or SEQ ID NO.20/13.

Preferably, the piRNA-54265 PolyA tailed PCR primer pair is shown as SEQID NO.8/21, SEQ ID NO.10/21, SEQ ID NO.11/22, SEQ ID NO.12/22, SEQ IDNO.14/23, SEQ ID NO.15/22, SEQ ID NO.16/22, SEQ ID NO.17/22, SEQ IDNO.18/22, SEQ ID NO.19/22 or SEQ ID NO.20/22.

Most preferably, the forward primer and the reverse primer of thestem-loop primer pair are respectively shown as SEQ ID NO.26 and SEQ IDNO.9.

Most preferably, the forward primer and the reverse primer of the PolyAtailed primer pair are respectively shown as SEQ ID NO.26 and SEQ IDNO.22.

Moreover, the kit further includes a probe used cooperatively with theprimers.

Preferably, the probe used cooperatively with the stem-loop primer isshown as SEQ ID NO.27.

In addition, the kit further includes an RNA reverse transcriptionprimer.

Preferably, the piRNA-54265 stem-loop reverse transcription primer has asequence shown as SEQ ID NO.2.

Preferably, the piRNA-54265 PolyA tailed reverse transcription primerhas a sequence shown as SEQ ID NO.3-5.

In addition, preferably, when using the kit of the present invention, ifa real-time fluorescent quantitative PCR is adopted, a reaction systemthereof is as shown in Table 13 and Table 14, and a reaction procedureis as shown in Table 15. If a droplet digital PCR is adopted, a reactionsystem thereof is as shown in Table 16, and a reaction procedure is asshown in Table 17.

The present invention has the following beneficial effects.

It is found and disclosed by the present invention for the first timethat there are statistically significant differences in expression ofthe piRNA-54265 in colorectal cancer and para-carcinoma tissues, and thepiRNA-54265 is high-expressed in cancer tissues. Therefore, thepiRNA-54265 can be used as a diagnostic marker for colorectal cancer andused for diagnosing colorectal cancer.

Meanwhile, it is also found by the present invention that the expressionlevel of the piRNA-54265 in the cancer tissues is related to thesurvival prognosis of the colorectal cancer patients, and the colorectalcancer patient with high expression level of piR-54265 has poorprognosis, so the piRNA-54265 can be used as a prognostic evaluationmarker for colorectal cancer and used for prognostic evaluation ofcolorectal cancer.

Furthermore, it is also founded by the present invention that theexpression of the piRNA-54265 can be detected in serums of thecolorectal cancer patients, and the expression level of the piRNA-54265in the serums is positively correlated with the expression level of thepiRNA-54265 in the cancer tissues, and is related to the survivalprognosis of the patients. In addition, the expression level of theserum piR-54265 of normal people is low, which is significantlydifferent and well differentiated in comparison with the expressionlevel of the serum piR-54265s of the colorectal cancer patients.Moreover, the serum piR-54265 level can effectively predict the onset ofcolorectal cancer, and the serum piR-54265 can be found elevated for upto 3 years before the diagnosis of cancer. Therefore, the piR-54265 cannot only be easily detected in a blood specimen, but also be used forperforming early diagnosis or preliminary screening. Moreover, it isalso founded by the present invention that with piRNA-54265 knockdown,the abilities of proliferation, migration and invasion of colorectalcancer cells are significantly weakened, and the growth and metastasisof xenograft tumors in nude mice transplanted with colorectal cancercells can also be significantly inhibited. Therefore, the piRNA-54265can be used as a therapeutic target for colorectal cancer and used fortreating colorectal cancer. Finally, it is also found by the presentinvention that when increasing or decreasing the expression level of thepiRNA-54265, the colorectal cancer cells show a certain degree ofreduction or enhancement of chemosensitivity; and the patients withhigh-expressed piRNA-54265 has poor short-term efficacy of chemotherapy.Therefore, the piRNA-54265 can be used as adjunctive therapy forchemotherapy.

The comprehensive results sufficiently show that the piR-54265 is animportant potential therapeutic target for cancer and a molecular markerfor cancer screening, diagnosis and treatment, but is not limited toserum, plasma or tissue, and is not limited to colorectal cancer. ThepiR-54265 is expected to be developed, transformed and applied inclinical work, and the effects thereof can be further explored andanalyzed in various systemic tumors.

Based on the above research results, the present invention furtherprovides a piRNA-54265 detection kit which can be used for earlyscreening, diagnosis, efficacy monitoring and prognosis evaluation ofcolorectal cancer. The present invention originally provides a specificprimer design pattern for the piRNA-54265, and verifies that both theamplification efficiency and specificity of the primers designed thereofare excellent. The kit provides a complete process from RNA purificationof specimen to the expression level detection of the target piRNA 54265.The process is already optimized for easy and efficient operation.Moreover, when the kit is in use, a specimen to be detected is suitablefor tissues, cells, cell supernatant and body fluids such as serum(plasma), urine, saliva, and other types of specimens with rare RNA, andthe required specimen amount is less than that of the same type of kitsin the market. Meanwhile, two optional expression detection methodsincluding real-time fluorescent quantitative PCR and absolutequantitative droplet digital PCR are provided, so the application rangeis wide.

BRIEF DESCRIPTION OF THE DRAWINGS

FIGS. 1a ˜1 d show that TCGA data analysis and tissue specimens ofpatients detection indicates the increased expression level of piR-54265in colorectal cancer.

FIG. 2 shows that high expression level of the piR-54265 in cancertissues associates with poor survival prognosis in colorectal cancerpatients.

FIGS. 3a ˜3 d show that high expression level of the piR-54265 in serumassociates with poor survival prognosis in colorectal cancer patients.

FIGS. 4a ˜4 d show the effects of piR-54265 expression on the capabilityof proliferation, colony formation, migration, invasion andanti-apoptosis in colorectal cancer cells.

FIGS. 5a ˜5 b show the effects of piR-54265 expression on subcutaneousxenograft growth and metastasis of CRC cells in nude mice.

FIGS. 6a ˜6 f show that piR-54265 bindsPIWIL2 promoting the activationof STAT3 and downstream signaling pathway.

FIGS. 7a ˜7 b show effects of piR-54265 on sensitivity of colorectalcancer cells and mouse subcutaneous xenograft tumors to chemotherapeuticagents.

FIGS. 8a ˜8 g show that a specific piR-54265 inhibitor inhibits thegrowth and metastasis of mouse subcutaneous xenografts.

FIGS. 9a ˜9 c show that high level of piR-54265 associates with a poorshort-term efficacy of chemotherapy in colorectal cancer patients.

FIG. 10 shows the piR-54265 level of the baseline serum in the normalpeople and people whom found onset of colorectal cancer in the laterfollow-up and grouped by the diagnosed time from blood sampling.

FIG. 11 shows schematic diagram of design modes for PCR primers of astem-loop primer method and a PolyA tailed method.

FIGS. 12a ˜12 b show amplification curves and melting curves of 11 pairsof primers of the stem-loop method.

FIGS. 13a ˜13 b show amplification curves and melting curves of 11 pairsof primers of the PolyA tailed method.

FIG. 14 shows amplification curves and melting curves of the optimalprimers of the stem-loop method and the PolyA tailed method.

FIGS. 15a ˜15 b show serum piRNA-54265 levels of one healthy control(normal people) during the follow-up visit via real-time fluorescencequantitative PCR detection of a kit, and one CRC case during thefollow-up visit (not found cancer at the time of sampling, before theonset of colorectal cancer), one sample with high-grade intraepithelialneoplasia in colorectum and two cases of colorectal cancer patients(stage I and IV).

FIGS. 16a ˜16 b show the serum piRNA-54265 levels of colorectal cancerpatients before operation, and in the first day, the third day, thefifth day and the seventh day after operation via the real-timefluorescence quantitative PCR detection of the kit; FIG. 16a showsamplification curves of the serum piRNA-54265 as well as externalreference cel-miR-39 of the samples in patients before and afteroperation; and FIG. 16b shows the corresponding melting curves.

FIG. 17 shows the serum piRNA-54265 levels in normal people andcolorectal cancer patients before treatment via digital PCR detection ofthe kit.

FIGS. 18a ˜18 d show the efficacy evaluation of the piRNA-54265detection kit.

DESCRIPTION OF THE EMBODIMENTS

The present invention is further described below with reference to theaccompanying drawings and specific embodiments, but the embodiments arenot intended to limit the present invention. Those of ordinary skills inthe art should understand that modifications or equivalent substitutionscan be made on the technical solutions of the present invention withoutdeparting from the spirit and scope of the technical solutions of thepresent invention. Any other changes, modifications, substitutions,combinations, and simplifications made without departing from the spiritand scope of the present invention are intended to be equivalents andare included in the protection scope of the present invention.

Unless otherwise indicated, the reagents, methods, and devices employedin the present invention are routine reagents, methods, and devices inthe art. The reagents and materials used in the following embodimentsare commercially available unless otherwise stated. Experimental methodsthat do not specify the specific conditions are usually in accordancewith conventional conditions, such as those described in textbooks andexperimental guides, or in accordance with the conditions recommended bythe manufacturers.

Embodiment 1 TCGA Data Analysis and Tissue Specimens of PatientsDetection Indicates the Increased Expression Level of a piR-54265 inColorectal Cancer

1. Experimental Method:

TCGA and GEO expression profile data were extracted and analyzed toobtain 20 top high-expressed piRNAs in colorectal cancer. Specificprimers were designed for the top 20piRNAs, and then expressionquantitation by qPCR was performed in 110 tumor tissue and pairednon-tumor tissue specimens of colorectal cancer patients from Guangzhou.The expression level of the identified piR-54265 was verified in another108 tumor tissue and paired non-tumor tissue specimens of colorectalcancer patients from Suzhou. Bioinformatics analysis of the gene copynumber variation and DNA methylation modification of piRNA-54265 wasperformed According to the COAD data in TCGA database.

2. Experimental Results:

As shown in FIGS. 1a and 1b , only the expressions of the piRNA-54265 inthe colorectal cancer tissue and the paired para-carcinoma tissue havesignificant statistical differences, the piRNA-54265 is high-expressedin the cancer tissue, and the same results are obtained from thespecimens of two clinical centers (Guangzhou and Suzhou).

The piRNA-54265 (Accession: DQ587153) is a mature body formed by aprecursor snoRNA57 thereof after intracellular processing, with asequence of 29 nucleotides in length and a genome located at CHR 20:2637585-2637613; and a sequence thereof is as follows:

SEQ ID NO. 29: tggaggtgatgaactgtctgagcctgacc, or SEQ ID NO. 30:UGGAG GUGAU GAACU GUCUG AGCCU GACC.

An effective sequence of the piRNA-54265, i.e., a reverse complementarysequence of the original sequence, is:

SEQ ID NO. 31: 5′-GGUCAGGCUCAGACAGUUCAUCACCUCCA-3′.

As shown in FIGS. 1c and 1d , DNA methylation and SCNA results suggestedthat the piRNA-54265 had reduced DNA methylation and increased gene copynumber amplification in colorectal cancer patients, and the differencewas statistically significant compared with a normal set. This furtherexplained the increased expression of the piRNA-54265 in colorectalcancer patients from the genomic level.

Embodiment 2 High Expression Level of the piR-54265 in Cancer TissuesAssociates with Poor Survival Prognosis in Colorectal Cancer Patients

1. Experimental Method:

SPSS (19.0) was used to analyze the expression of the piR-54265 incancer tissues and the prognosis of colorectal cancer patients (such asthe above-mentioned 110 colorectal cancer patients with postoperativechemotherapy from Guangzhou and 108 colorectal cancer patients withpostoperative chemotherapy from Suzhou).

2. Experimental Results:

The expression level of the piR-54265 in cancer tissues was related tothe survival prognosis of the colorectal cancer patients. Thehigh-expressed piR-54265 was associated with poor prognosis of thecolorectal cancer patients, having both shorter 5-year overall survivaland 5-year progression-free survival. The results were consistent in thetwo sample sets, Guangzhou and Suzhou (as shown in FIG. 2).

Embodiment 3 High Expression Level of the piR-54265 in Serum Associateswith Poor Survival Prognosis of in Colorectal Cancer Patients

1. Experimental Method:

four serum specimens were randomly selected from the samples ofcolorectal cancer cases previously mentioned for Northern Blot todirectly determine the presence or absence of piR-54265 in serum; then,RNA extraction and piR-54265 expression detection by RT-qPCR wereperformed on all serum specimens, and the correlation between the serumpiR-54265 and the piR-54265 level in the cancer tissues was analyzed.Survival analysis was performed on the serum piR-54265 level and theprognosis of the corresponding patients. RNA was extracted from 111serum specimens of normal people followed by piR-54265 expressiondetection via RT-qPCR and unpaired Student's t-test statistical analysiswas performed to compare the serum piR-54265 levels of normal people andthe colorectal cancer patients.

2. Experimental Results:

As shown in FIG. 3a , the piRNA-54265 could be detected in the serum ofthe colorectal cancer patients. In FIG. 3b and FIG. 3c , the expressionlevel of the piRNA-54265 in the serums of the patients was positivelycorrelated with the expression level of the piRNA-54265 in the cancertissues, and was negatively related to the survival prognosis of thepatients. In FIG. 3d , the expression level of the serum piR-54265 inthe colorectal cancer patients was higher than that in the normalpeople, and the difference was statistically significant. The aboveresults indicate that the expression level of the serum piR-54265 hasthe potential as an important marker for early diagnosis and prognosisprediction of colorectal cancer.

Embodiment 4 Effects of piR-54265 Expression on the Capability ofProliferation, Colony Formation, Migration, Invasion and Anti-Apoptosisin Colorectal Cancer Cells

1. Experimental Method:

Colorectal cancer cell lines HCT116 and LoVo with stably overexpressedand knockdown piR-54265 were successfully constructed, and then colonyformation, cell migration or invasion, CCK-8 cell viability detection,cell apoptosis by Annexin-V/PI flow cytometry and cell cycle analysis byflow cytometry were performed on these cells.

2. Experimental Results:

As shown in FIG. 4, functional verification of the piRNA-54265 wasperformed in two colorectal cancer cell lines in vitro, and the resultsdemonstrated that: compared with the control, overexpression of thepiRNA-54265 significantly enhanced the ability of proliferation (FIG. 4a), colony formation (FIG. 4b ), anti-apoptosis (FIG. 4c ) as well asinvasion and migration (FIG. 4d ) of the colorectal cancer cells. On thecontrary, after knocking down the piRNA-54265, both the proliferationand invasion abilities of the colorectal cancer cells were significantlydecreased. perturbing the expression of the piR-54265 did not affect theprogression of cell cycle (not shown).

Embodiment 5 Effects of piR-54265 Expression on Subcutaneous XenograftGrowth and Metastasis of CRC Cells in Nude Mice

1. Experimental Method:

Colorectal cancer cell lines with stably overexpressed (OE) or knockdown(KD) piR-54265 were inoculated subcutaneously into nude mice to formatsubcutaneous xenograft tumors. The volume of the subcutaneous xenografttumors was recorded weekly after successful modeling. The piR-54265 OEand KD cell lines were further constructed to co-express luciferase(Luc). Luc cells were injected into the nude mice via tail vein toestablish metastatic xenograft tumors. After successful modeling, anIn-vitro Imaging System (IVIS) was used to periodically detect Lucphoton values in the nude mice to monitor the progress of themetastasis.

2. Experimental Results:

As shown in FIGS. 5a-5b , the results of the animal experimentsdemonstrated that, the overexpression of the piRNA-54265 could promotethe growth of the subcutaneous xenograft tumors as well as distantmetastasis and colonization of the cancer cells in the nude mice; theresults were reversed while the piRNA-54265 was knockdown.

Embodiment 6 Study on the Molecular Mechanism of piR-54265 in PromotingGeneration and Development of Colorectal Cancer

1. Experimental Method:

In an RNA immunoprecipitation (RIP) experiment, an antibody of a proteinin PIWI protein family was used to perform immunoprecipitation (IP) intotal RNA of colorectal cancer cells, and RT-qPCR was then performedafter the PIWI protein precipitated RNAs were eluted. In a Pull Downexperiment, the synthesized biotin-labeled piR-54265 was incubated withstreptomycin magnetic beads and then incubated in fresh protein lysatesof HCT116 and LoVo cells respectively, and then Western Blot wasperformed after the piR-54265-pulldownproteins were eluted. Western Blotwas performed to analyze the changes of the expression of the downstreamproteins when perturbing the expression of the piR-54265. In a Co-IPexperiment, fresh protein lysates of cells with perturbed piR-54265 wereincubated with PIWIL2 or STAT3 antibody respectively followed by WesternBlot to analyze the combination of PIWIL2 and STAT3.

2. Experimental Results:

FIG. 6a and FIG. 6b endogenously and exogenously proved that thepiR-54265 can specifically bind with the PIWIL2 protein. Perturbing theexpression level of the piR-54265 do not obviously change the levels ofSTAT3 and SRC proteins but the phosphorylated STAT3 (p-STAT3). Theexpression of the p-STAT3 protein was up-regulated when overexpressingthe piR-54265 (FIG. 6c ).

Furthermore, the Co-IP experiment demonstrated that the PIWIL2, STAT3and SRC protein could combine with each other, and the overexpression orknockdown of the piR-54265 could increase or reduce their combination(FIG. 6d ).

Western blot analysis verified that, piR-54265 interacts with STAT3 andregulates a series of apoptosis and metastasis related downstreammolecules of STAT3 (FIG. 6e ).

In this study, a series of molecular biology experiments were used toelucidate a cancer-promoting mechanism of the piRNA-54265 (signalingpathway and molecular mechanism shown in FIG. 60, which could bindPIWIL2, and then promote the activation of STAT3 and its downstreamanti-apoptosis and metastasis signaling, thus promoting the growth andmetastasis of colorectal cancer.

Embodiment 7 Effects of piR-54265 on Drug Sensitivity of ColorectalCancer Cells

1. Experimental Method:

IC₅₀ experiments with the first-line colorectal cancer chemotherapydrugs 5-FU and L-OHP were respectively performed on colorectal cancercell lines with stably overexpressed or knockdown piR-54265.Subcutaneous xenograft tumors in nude mice were established using thestable cell lines mentioned above, and when a tumor volume reached 250mm³, treatment of intraperitoneal injection with 5-FU or L-OHP wasstarted, and the change of the tumor volume was periodically measured.

2. Experimental results: as shown in FIG. 7a , the changes of the IC₅₀of the colorectal cells suggested that piRNA-54265 could affect thechemosensitivity of colorectal cancer cells. When overexpressing thepiR-54265, the chemoresistance of the colorectal cancer cells to 5-FUand L-OHP could be enhanced, and the cell chemosensitivity was improvedafter knocking down the piR-54265. The results were consistent in theanimal experiments (FIG. 7b ): overexpression of the piR-54265significantly reduced the efficacy of chemotherapy drugs on thesubcutaneous xenograft tumors, while knockdown of piR-54265significantly enhanced the chemosensitivity to the drugs and thexenograft tumors shrank remarkably.

Embodiment 8 Specific piR-54265 Inhibitor Inhibits the Growth andMetastasis of Mouse Subcutaneous Xenografts

1. Experimental Method:

Colorectal cancer cell lines with stably overexpressed or knockdownpiR-54265 were inoculated subcutaneously into a nude mouse respectivelyto establish subcutaneous xenograft tumors. When the tumor volumereached 50 mm³, a specific piR-54265 inhibitor was multi-pointedlyinjected into the tumors, and the tumor volume was monitored andrecorded regularly (see FIG. 8a for specific operation time). ThepiR-54265 overexpressed (OE) or knockdown (KD) colorectal cancer cellsco-expressing Luciferase were injected into the nude mice via tail veinto establish metastatic xenograft tumors, and the luminescence value wasmonitored by IVIS regularly. When the luminescence value reached1×10⁶p/sec/cm²/sr, the specific piR-54265 inhibitor was injected viatail vein or combined with chemotherapy drug 5-FU intraperitoneallyinjected periodically, and the progress of the metastasis was monitoredby IVIS (see FIG. 8b for specific operation time).

2. Experimental Results:

The results were shown in FIGS. 8c to 8g . Using the specificpiRNA-54265 inhibitor or combined with chemotherapy drug for tumortreatment in nude mice could effectively inhibit the growth of thesubcutaneous and metastatic xenograft tumors, and significantlyprolonged the overall survival time of the metastatic xenograft nudemice. Therefore, the specific piR-54265 inhibitor had a certainanti-cancer effect and could assist in improving curative effect of thechemotherapy drugs, and the piR-54265 inhibitor might probably become anew therapeutic target for colorectal cancer.

Embodiment 9 High Level of piR-54265 Associates with a Poor Short-TermEfficacy of Chemotherapy in Colorectal Cancer Patients

1. Experimental Method:

Serum specimens were collected before any treatment from colorectalcancer patients who received preoperative neoadjuvant chemotherapy inGuangzhou (215 cases, training set) and Beijing (102 cases, verificationset). Total RNAs were extracted from the serum samples followed byRT-qPCR detection for piRNA-54265 expression. Then analyze therelationship between the piR-54265 expression of the patients and theircurative efficacy after neoadjuvant chemotherapy, and ROC curve wasperformed to evaluate the efficacy of serum piR-54265 in predictingchemotherapeutic effect and obtain an optimal cutoff value.

2. Experimental Results:

In order to more directly investigate the relationship between thepiRNA-54265 and chemotherapy, and eliminate the interference of manycomplicated factors i.e. operation, serum specimens were selected beforetreatment from colorectal cancer patients who received neoadjuvanttherapy. As shown in FIG. 9a and FIG. 9b , patients with high-expressedpiRNA-54265 in serum had poor short-term efficacy of chemotherapy; andthe results in two sample sets were consistent. The ROC curve wasconstructed for the short-term efficacy of chemotherapy of patientsbased on the expression level of piR-54265 in serum, and the resultswere shown in FIG. 9c . In the training set, the expression level of thepiR-54265 in serum could accurately reflect the efficacy of chemotherapyor chemosensitivity of the colorectal cancer patients (AUC=0.819,P<0.0001, Guangzhou), and the corresponding cutoff value of the ROCcurve was 0.01227 (the sensitivity was 66.7%, and the specificity was90.0%). The Cutoff value was used to model and simulate in theverification set, wherein the sensitivity was 53.3%, the specificity was93.1%, AUC=0.808, and P<0.0001. Therefore, the piRNA-54265 would be apotential and promising specific molecular marker of colorectal cancer,which could be easily detected in blood specimen and specificallyindicated the chemosensitivity and prognosis of the colorectal cancerpatients, and the expression level of the piR-54265 in serum and thecutoff value would be helpful for more accurate individualized selectionof clinical therapeutic regime and improvement of prognosis.

Embodiment 10 Prospective Nested Case-Control Study on the Efficacy ofBaseline Serum piR-54265 Level in Early Screening for Colorectal Cancerin General Population

1. Experimental Method:

A total of 1160 baseline serum specimens were collected from 2008 to2013 from normal people whom were followed-up for a long time to monitorand record the cancer status. Under the double-blind design, theexpression of piR-54265 was absolutely quantitated in the 1160 serumspecimens from the normal people by droplet digital PCR. Afterunblinding, statistical analysis was performed to investigate theefficacy of the baseline serumpiR-54265 level in predicting the risk ofcolorectal cancer of general people or recognizing colorectal cancerfrom general people in advance.

2. Experimental Results:

The results were shown in FIG. 10. The closer to the time of finaldiagnosis of colorectal cancer, the higher the expression level of theserum piR-54265 was. The serum piR-54265 could effectively predict theonset of colorectal cancer, and the serum piR-54265 could be detectedsensitively high-expressed up to 3 years before the onset of cancer.

Therefore, the serum piR-54265 could be used for early screening andearly diagnosis of colorectal cancer in general people, which couldeffectively predict the onset three years earlier.

In summary, the research results showed that: the high-expressedpiRNA-54265 (piR-54265) in colorectal cancer tissues and serums/plasmapromoted malignant growth and metastasis of colorectal cancer cells. ThepiR-54265 was closely related to the onset, prognosis andchemotherapeutic efficacy of the colorectal cancer, was a potentialevaluation marker for early screening, early diagnosis, prognosticevaluation and chemosensitivity of the colorectal cancer, and was apotential therapeutic target for colorectal cancer. We further detectedthe serum piR-54265 levels in several common digestive system tumors,and the results showed the specificity and high expression of the serumpiR-54265 in colorectal cancer. In a prospective study for screeningcolorectal cancer in a large cohort of general people, the resultsshowed that the serum piR-54265 could effectively predict the onset ofcolorectal cancer, which was three years earlier than the diagnosedtime.

The comprehensive results sufficiently showed that the piR-54265 was animportant and potential therapeutic target for colorectal cancer and amolecular marker for screening, diagnosis and treatment of colorectalcancer, and was not limited to the type of specimen, serums, plasma ortissues, and was not limited to colorectal cancer either. The piR-54265was expected to be developed, transformed and applied in clinical work,and the effects thereof could be further explored and analyzed invarious systemic tumors.

Therefore, early screening, early diagnosis, as well as chemosensitivityand prognosis evaluation of the colorectal cancer could be performed bydetecting the expression level of the piR-54265, and gene therapy forcolorectal cancer could be performed using the piR-54265 as a target.

The following embodiments 11 to 15 present a solution suitable fordetecting the expression level of the piR-54265, including design ofdetection primers, a detection method and construction of a detectionkit, and evaluation of the kit.

Embodiment 11 piR-54265 Primer Design and Specific Design Model

1. Reverse transcription primers (as shown in Table 1 and Table 2) wererespectively designed by two small RNA reverse transcription methodsincluding a stem-loop method and a PolyA tailed method, and PCR primerswere designed accordingly so as to quantify the piRNA-54265. Thespecific design ideas were shown in FIG. 11 below.

Design principles for forward primers of the stem-loop method and thePolyA tailed method were similar. A forward primer is designed based onthe sequence of the piRNA-54265 (which changes U in the RNA sequence ofthe piRNA-54265 to T), and sequentially includes an amount of bases fromthe 5′⁻ of the sequence, and shall include the first five bases “TGGAG”at least. In general, fluorescence quantitative PCR primers were 17 to25 nt in length. Therefore, when the current primer was designed to be afull length of the piRNA-54265 theoretically, which was 29 nt (like aforward primer 1 in Table 3 below), the cDNA of the piRNA-54265 formedby reverse transcription could also be specifically amplified. On theother hand, when appropriately reducing the length of the forwardprimers, i.e., reducing the number of bases of the forward primerscomplementary with the cDNA, the forward primers could also bequantitatively amplified, but when the complementary bases were reducedto a certain number, the forward primers could not be effectivelyamplified. Therefore, under the premise of ensuring efficientamplification, we discussed the minimum number of the bases of theforward primers complementary with the cDNA. The numbers of the bases ofthe designed forward primers complementary with a terminal of a cDNA3′was sequentially decreased, which were 29, 25, 21, 17, 13, 9, 5, 4, 3, 2and 1 respectively. The primer sequences were shown in Table 3 and Table4 below. Corresponding amplification curves and melting curves wereshown in FIGS. 12a-12b and FIGS. 13a-13b . It could be seen that theforward primer of the piRNA-54265 could be effectively amplified fromthe last base at the terminal of 3, from 3′ to 5′ when the basescontinuously complementary with the terminal of the cDNA3′ were no lessthan 5 bases (TGGAG), and could not be effectively amplified when thecomplementary bases were 4, 3, 2, and 1 (i.e., the primer pairs in Table3 and Table 4, and the primer pairs without TGGAG in the forward primerscould not be effectively amplified).

TABLE 1 piRNA-54265 reverse transcription primer Sequence Serial No.Note Sequence length (nt) SEQ ID NO. 1 piRNA-54265TGGAGGTGATGAACTGTCTGAG 29 CCTGACC SEQ ID NO. 2 piRNA-54265TGACCGTCTGTATGGTTGTTCAC 67 stem-loop reverse GACTCCTTCACCCTATCCAACCAtranscription TACAGACGGTCAGGTCAGGCT primer SEQ ID NO. 3 piRNA-54265GCTGTCAACGATACGCTACGTA 61 PolyA tailed ACGGCATGACAGTGT(24)A SEQ ID NO. 4reverse GCTGTCAACGATACGCTACGTA 61 transcription ACGGCATGACAGTGT(24)GSEQ ID NO. 5 primer GCTGTCAACGATACGCTACGTA 61 ACGGCATGACAGTGT(24)C

TABLE 2 External reference cel-miR-39reverse transcription primerSequence Serial No. Note Sequence length (nt) SEQ ID NO. 6cel-miR-39sequence TCACCGGGTGTAAATCAGCTTG 22 SEQ ID NO. 7cel-miR-39stem- TGAACATCCTCTGGAGGCCAAC 76 loop reverseTGCGTGAGCTTGTTACTCATTTT transcription CTCAGCCTCCAGAGGATGTTCAC primerAAGCTGAT SEQ ID NO. 3 cel-miR-39PolyA GCTGTCAACGATACGCTACGTA 61tailed reverse ACGGCATGACAGTGT(24)A SEQ ID NO. 4 transcriptionGCTGTCAACGATACGCTACGTA 61 primer ACGGCATGACAGTGT(24)G SEQ ID NO. 5GCTGTCAACGATACGCTACGTA 61 ACGGCATGACAGTGT(24)C

TABLE 3 piRNA-54265 stem-loop PCR primer Sequence Serial No. NoteSequence length (nt) SEQ ID NO. 8 piRNA-54265 stem-loopTGGAGGTGATGAACTGT 29 forward primer 1 CTGAGCCTGACC SEQ ID NO. 9piRNA-54265 stem-loop TATGGTTGTTCACGACT 24 reverse primer 1 CCTTCACSEQ ID NO. 10 piRNA-54265 stem-loop TGGAGGTGATGAACTGT 25forward primer 2 CTGAGCCT SEQ ID NO. 9 piRNA-54265 stem-loopTATGGTTGTTCACGACT 24 reverse primer 2 CCTTCAC SEQ ID NO. 11piRNA-54265 stem-loop TGGAGGTGATGAACTGT 21 forward primer 3 CTGASEQ ID NO. 9 piRNA-54265 stem-loop TATGGTTGTTCACGACT 24 reverse primer 3CCTTCAC SEQ ID NO. 12 piRNA-54265 stem-loop TGGAGGTGATGAACTGT 17forward primer 4 SEQ ID NO. 13 piRNA-54265 stem-loop TATGGTTGTTCACGACT17 reverse primer 4 SEQ ID NO. 14 piRNA-54265 stem-loop TGGAGGTGATGAA 13forward primer 5 SEQ ID NO. 13 piRNA-54265 stem-loop TATGGTTGTTCACGACT17 reverse primer 5 SEQ ID NO. 15 piRNA-54265 stem-loopCTATCGCATGCTGGAGG 20 forward primer 6 TGA SEQ ID NO. 13piRNA-54265 stem-loop TATGGTTGTTCACGACT 17 reverse primer 6SEQ ID NO. 16 piRNA-54265 stem-loop TCGACTATCGCATGCTG 20forward primer 7 GAG SEQ ID NO. 13 piRNA-54265 stem-loopTATGGTTGTTCACGACT 17 reverse primer 7 SEQ ID NO. 17piRNA-54265 stem-loop TCGACTATCGCATGCTG 19 forward primer 8 GASEQ ID NO. 13 piRNA-54265 stem-loop TATGGTTGTTCACGACT 17reverse primer 8 SEQ ID NO. 18 piRNA-54265 stem-loop TCGACTATCGCATGCTG18 forward primer 9 G SEQ ID NO. 13 piRNA-54265 stem-loopTATGGTTGTTCACGACT 17 reverse primer 9 SEQ ID NO. 19piRNA-54265 stem-loop TCGACTATCGCATGCTG 17 forward primer 10SEQ ID NO. 13 piRNA-54265 stem-loop TATGGTTGTTCACGACT 17reverse primer 10 SEQ ID NO. 20 piRNA-54265 stem-loop TCGACTATCGCATGCT16 forward primer 11 SEQ ID NO. 13 piRNA-54265 stem-loopTATGGTTGTTCACGACT 17 reverse primer 11

TABLE 4 piRNA-54265 PolyA tailed PCR primer Sequence Serial No. NoteSequence length (nt) SEQ ID NO. 8 piRNA-54265 PolyA tailedTGGAGGTGATGAACTG 29 forward primer 1 TCTGAGCCTGACC SEQ ID NO. 21piRNA-54265 PolyA tailed GCTGTCAACGATACGC 25 reverse primer 1 TACGTAACGSEQ ID NO. 10 piRNA-54265 PolyA tailed TGGAGGTGATGAACTG 25forward primer 2 TCTGAGCCT SEQ ID NO. 21 piRNA-54265 PolyA tailedGCTGTCAACGATACGC 25 reverse primer 2 TACGTAACG SEQ ID NO. 11piRNA-54265 PolyA tailed TGGAGGTGATGAACTG 21 forward primer 3 TCTGASEQ ID NO. 22 piRNA-54265 PolyA tailed GCTGTCAACGATACGC 20reverse primer 3 TACG SEQ ID NO. 12 piRNA-54265 PolyA tailedTGGAGGTGATGAACTG 17 forward primer 4 T SEQ ID NO. 22piRNA-54265 PolyA tailed GCTGTCAACGATACGC 20 reverse primer 4 TACGSEQ ID NO. 14 piRNA-54265 PolyA tailed TGGAGGTGATGAA 13 forward primer 5SEQ ID NO. 23 piRNA-54265 PolyA tailed GCTGTCAACGATACG 15reverse primer 5 SEQ ID NO. 15 piRNA-54265 PolyA tailed CTATCGCATGCTGGAG20 forward primer 6 GTGA SEQ ID NO. 22 piRNA-54265 PolyA tailedGCTGTCAACGATACGC 20 reverse primer 6 TACG SEQ ID NO. 16piRNA-54265 PolyA tailed TCGACTATCGCATGCTG 20 forward primer 7 GAGSEQ ID NO. 22 piRNA-54265 PolyA tailed GCTGTCAACGATACGC 20reverse primer 7 TACG SEQ ID NO. 17 piRNA-54265 PolyA tailedTCGACTATCGCATGCTG 19 forward primer 8 GA SEQ ID NO. 22piRNA-54265 PolyA tailed GCTGTCAACGATACGC 20 reverse primer 8 TACGSEQ ID NO. 18 piRNA-54265 PolyA tailed TCGACTATCGCATGCTG 18forward primer 9 G SEQ ID NO. 22 piRNA-54265 PolyA tailedGCTGTCAACGATACGC 20 reverse primer 9 TACG SEQ ID NO. 19piRNA-54265 PolyA tailed TCGACTATCGCATGCTG 17 forward primer 10SEQ ID NO. 22 piRNA-54265 PolyA tailed GCTGTCAACGATACGC 20reverse primer 10 TACG SEQ ID NO. 20 piRNA-54265 PolyA tailedTCGACTATCGCATGCT 16 forward primer 11 SEQ ID NO. 22piRNA-54265 PolyA tailed GCTGTCAACGATACGC 20 reverse primer 11 TACG

TABLE 5 External reference cel-miR-39 stem-loop PCR primer SequenceSerial No. Note Sequence length (nt) SEQ ID NO. 24 cel-miR-39stem-CGGCTCACCGGGTGTAAATC 20 loop forward primer SEQ ID NO. 25cel-miR-39stem- CAACTGCGTGAGCTTGTTACTC 22 loop reverse primer

TABLE 6 External reference cel-miR-39PolyA tailed PCR primer SequenceSerial No. Note Sequence length (nt) SEQ ID NO. 24 cel-miR-39PolyACGGCTCACCGGGTGTAAATC 20 tailed forward primer SEQ ID NO. 22cel-miR-39PolyA GCTGTCAACGATACGCTACG 20 tailed reverse primer

2. Further Optimization of Primers:

According to the results of FIGS. 12 and 13, the amplificationefficiencies of the third stem-loop primer pair (i.e., corresponding toSEQ ID NO.11&9) and the fourth PolyA tailed primer pair (i.e.,corresponding to SEQ ID NO.11&22) were higher than that of otherprimers, so these two pairs of primers were selected as candidateprimers with higher preference.

Based on the candidate optimal primers screened above, C and G wereintroduced at the 5′ end of the primer for further optimization. Theoptimized primer sequences were shown in Tables 7 and 8 below, and thecorresponding amplification curves and melting curves were shown in FIG.14.

TABLE 7 Optimal stem-loop primer pair Sequence Serial No. Note Sequencelength (nt) SEQ ID NO. 26 piRNA-54265 CCTGGAGGTGATGAACTGTCT 22stem-loop forward G primer 12 SEQ ID NO. 9 piRNA-54265TATGGTTGTTCACGACTCCTT 24 stem-loop reverse CAC primer 12

TABLE 8 Optimal PolyA tailed primer pair Sequence Serial No. NoteSequence length (nt) SEQ ID NO. 26 piRNA-54265 PolyACCTGGAGGTGATGAACTGTC 22 tailed forward TG primer 12 SEQ ID NO. 22piRNA-54265 PolyA GCTGTCAACGATACGCTACG 20 tailed reverse primer 12

3. Digital PCR platform detection involved the use of probes,corresponding probes were designed according to the optimal primer, asshown in Table 9:

TABLE 9 Stem-loop probe Sequence Serial No. Note Sequence length (nt)SEQ ID NO. 27 piRNA-54265stem-loop FAM-CCCTATCCAACCATA 27 probeCAGACGGTCAGG-BHQ1 SEQ ID NO. 28 cel-miR-39 stem-loop HEX-ATTTTCTCAGCCTCC27 probe AGAGGATGTTCA-BHQ1

Embodiment 12 piRNA-54265 Detection Method

1. Total RNA Extraction from Specimen:

(1) Total RNA extraction from serum:

1) 100 μl of plasma (serum) was added to a reaction plate.

2) A proteinase K digestion, wherein a digestion system was: 45 μl ofproteinase K digestion buffer, 5 μl of proteinase K (50 mg/ml), and atotal volume of 50 μl.

3) A mixture was shaken up and incubated for 5 minutes on a vortexer;incubated for 30 minutes at 65° C.; and then shaken up and incubated for5 minutes on the vortexer at 700 rpm.

4) A lysis &RNA binding system (Lysis Binding Mix) was prepared: 99 μlof lysis buffer, 1 μl of 2-mercaptoethanol, and a total volume of 100μl.

5) 100 μl of the Lysis Binding Mix, 5 fmol of external referencecel-miR-39, and 20 μl of magnetic beads (RNA Binding Beads) weresequentially added.

6) The mixture was shaken up and incubated for 7 minutes on the vortexerat 400 rpm.

7) The mixture was eluted by isopropanol, shaken up and incubated for 15minutes on the vortexer.

8) The mixture was reversed and blended again like step 7).

9) The reaction plate was placed on a magnetic stand until the solutionwas clear; and a supernatant was discarded.

10) 180 μl of complete eluent 1 (complete eluent 1:10 ml of isopropanolwas added into an eluent 1) was added and the reaction plate was shakenup on the vortexer for 1 minute at 700 rpm.

11) The reaction plate was placed on the magnetic stand for 1 minuteuntil the solution became clear; and a supernatant was discarded.

12) Steps 10) and 11) were repeated with 180 μl of complete eluent 2(complete eluent 2: 48 ml of absolute ethyl alcohol was added in aneluent 2).

13) The mixture was placed on the vortexer and spin-dried for 5 minutes.

14) DNAzymes was added for digestion, wherein a digestion system was: 48μl of DNase buffer, 2 μl of DNase (20 U/μl), and a total volume of 50μl.

15) After being film-pasted, the mixture was placed on the vortexer andshaken up for 15 minutes.

16) 50 μl of rebinding buffer and 100 μl of isopropanol wererespectively added successively. The mixture was shaken up for 5 minuteson the vortexer.

17) The reaction plate was placed on the magnetic stand for 5 minutesuntil the solution became clear; and a supernatant was discarded.

18) 180 μl of complete eluent 2 was added and film-pasted, and thereaction plate was shaken up on the vortexer for 30 seconds at 700 rpm.

19) The reaction plate was placed on the magnetic stand for 1 minuteuntil the solution became clear; and a supernatant was discarded.

20) Steps 18) and 19) were repeated.

21) The mixture was air-dried on the vortexer for 5 minutes.

22) 50 μl of enzyme-free water (Elution Buffer) preheated to 65° C. wasadded, and then the reaction plate was shaken up on the vortexer for 2minutes.

23) The reaction plate was placed in a water bath at 65° C. forincubation for 5 minutes.

24) The reaction plate was placed on the magnetic stand until thesolution became clear; and a supernatant (extracted RNA) was transferredto an enzyme-free tube and stored at −80° C.

(2) Total RNA Extraction from Urine Specimen:

1) 50 μl of urine specimen was added into a reaction plate.

2) A lysis &RNA binding system (Lysis Binding Mix) (one specimen hole)was prepared: 198 μl of lysis buffer, 2 μl of (3-mercaptoethanol, and atotal volume of 200 μl.

3) 200 μl of Lysis Binding Mix was added into the reaction plate in step2), and a final volume was adjusted to 450 μl, then the reaction platewas sealed with a film, and shaken up and blended on a vortexer for 7minutes at 300 rpm.

4) 30 μl of magnetic bead binding system was added into the reactionplate, wherein the magnetic bead binding system (one specimen hole) was:20 μl of RNA binding magnetic beads, 10 μl of lysis/binding promotingsolution, and a total volume of 30 μl.

5) The reaction plate was sealed with a film, shaken up and blended onthe vortexer for 5 minutes at 300 rpm.

6) The mixture was eluted by isopropanol, then shaken up and blended for20 minutes on the vortexer.

7) The reaction plate was placed on a magnetic stand until the solutionwas clear; and a supernatant was discarded.

8) 180 μl of complete eluent 1 was added, and the reaction plate wassealed with a film and blended on the vortexer for 1 minute at 700 rpm.

9) The reaction plate was placed on the magnetic stand until thesolution became clear; and a supernatant was discarded.

10) Steps 8) and 9) were repeated with a complete eluent 2.

11) The mixture was spin-dried on the vortexer for 5 minutes.

12) DNAzymes was added for digestion, and a digestion system was: 48 μlof DNase buffer, 2 μl of DNase (20 U/μl), and a total volume of 50 μl,sealed with a film, and then shaken up for incubation for 15 minutes;

13) 50 μl of rebinding buffer and 100 μl of isopropanol weresequentially added into the reaction plate.

14) The reaction plate was sealed with a film, and shaken up for 5minutes on the vortexer.

15) The reaction plate was placed on the magnetic stand until thereaction solution became clear, and a supernatant was discarded.

16) 180 μl of complete eluent 2 was added, and the reaction plate wasshaken up on the vortexer for 30 seconds.

17) The reaction plate was placed on the magnetic stand until thereaction solution became clear; and a supernatant was discarded.

18) Steps 16) and 17) were repeated with the complete eluent 2.

19) The reaction plate was spin-dried on the vortexer for 5 minutes.

20) 50 μl of enzyme-free water preheated to 65° C. was added, then thereaction plate was sealed with a film and placed on the vortexer forincubation for 2 minutes.

21) The reaction plate was put into a water bath for incubation at 65°C. for 5 minutes.

22) The reaction plate was placed on the vortexer for incubation for 2minutes.

23) The reaction plate was placed on the magnetic stand until thesolution became clear; and a supernatant was transferred to anenzyme-free tube and stored at −80° C.

2. Reverse Transcription:

(1) First-strand synthesis of cDNA by stem-loop reverse transcription:

1) A reverse transcription system was prepared as shown in Table 10below (for each specimen):

TABLE 10 Stem-loop reverse transcription system Constituent Volume (μl)5 × reverse transcription reaction buffer^([1]) 4 (5 × Reaction Buffer)10 mM deoxyribonucleotide mixture^([2]) (10 mM dNTPMix) 2 Moloney murineleukemia virus reverse transcriptase 1 with low H activity of RNase (200U/μl RevertAid ™ M-MuLV Reverse Transcriptase) RNase inhibitor (20 U/μlRNase Inhibitor) 1 piRNA-54265 reverse transcription primer (10 pmol/μl)1 cel-miR-39 reverse transcription primer (10 pmol/μl) 1 Reversetranscription system 10 Remarks: [1] The 5 × reverse transcriptionreaction buffer was formulated by tris(hydroxymethyl)aminomethane, i.e.,Tris-HCl (pH = 8.3), potassium chloride (KCl), magnesium chloride(MgCl₂), and dithiothreitol (DTT). [2] 10 mM deoxyribonucleotide mixturecontained four deoxyribonucleotides dATP, dCTP, dGTP and dTTP at aconcentration of 10 mM, and was dissolved in 0.6 mM Tris-HCl buffer (pH= 7.5).

2) 10 μl of RNA was extracted from the 50 μl of total RNA extracted, andthen added into 10 μl of the reverse transcription system above, anddetached. The mixture was placed in a temperature cycler for reversetranscription: 60 minutes at 42° C., and 5 minutes at 70° C.

3) The first-strand of the cDNA synthesized by reverse transcriptioncould be placed on ice for standby service or stored at −20° C. for along time.

(2) First-strand synthesis of cDNA by PolyA tailed reversetranscription:

1) A PolyA tail was added to RNA, and a tail was added to the RNA in thespecimen according to a reaction system in Table 11 below, and incubatedat 37° C. for 10 minutes.

TABLE 11 Constituent Volume (μl) Polyadenine nucleotide polymerase (5000U/ml) 1 10 × polyadenine nucleotide polymerization reaction buffer 2RNase inhibitor 1 10 mM ATP 2 RNA 1-10 μg RNase-free water made up to avolume of 20 μl Total volume 20 

2) First-strand synthesis of cDNA by reverse transcription:

Reverse transcription was performed on the RNA already added with thePolyA tail in 1), and a reverse transcription reaction system wasconfigured as shown in Table 12 below.

TABLE 12 Volume Constituent (μl) RNA added with PolyA tail 10 5 ×reverse transcription reaction buffer (5 × Reaction Buffer) 4 10 mMdeoxyribonucleotide mixture (10 mM dNTP Mix) 2 Moloney murine leukemiavirus reverse transcriptase with low H 1 activity of RNase (200 U/μlRevertAid ™ M-MuLV Reverse Transcriptase) RNase inhibitor (20 U/μl RNaseInhibitor) 1 Reverse transcription primer mixture (10 pmol/μl) 1Enzyme-free water 1 Total volume 20

3. PCR detection:

(1) Real-time fluorescence quantitative PCR:

1) Reaction system:

cDNA stock solution was appropriately diluted and subjected toquantitative PCR. The target piRNA, external references and reactionsystem were shown in Tables 13 and 14 below, with 2 to 3 repetitionrespectively:

TABLE 13 Volume Final Constituent (μl) concentration 2 × SYBR GREEN 5 1×piRNA-54265 forward primer (10 pmol/μl) 1 1 pmol/μl piRNA-52465reverseprimer (10 pmol/μl) 1 1 pmol/μl Water 2 — cDNA of appropriate dilutionratio 1 — Quantitative PCR system 10 —

TABLE 14 Final Constituent Volume (μl) concentration 2 × SYBR GREEN 5 1×cel-miR-39 forward primer (10 pmol/μl) 1 1 pmol/μl cel-miR-39 reverseprimer (10 pmol/μl) 1 1 pmol/μl Water 2 — cDNA of appropriate dilutionratio 1 — Quantitative PCR system 10 —

2) PCR Amplification Reaction:

9 μl buffer of the reaction system was added into each hole of the PCRreaction plate, and then 1 μlcDNA of appropriate dilution ratio wasadded to perform the amplification reaction according to Table 15 below.

TABLE 15 Step Temperature Time Cycle Pre-denaturizing 95° C.  5 minutes1 Amplifying Denaturizing 95° C. 10 seconds 45 Annealing 60° C. 10seconds Extending 72° C. 10 seconds Melting curve 95° C.  5 seconds 165° C.  1 minute 97° C. — Cooling 40° C. 30 seconds 1

3) Analysis of Results:

The PCR amplification results were represented by CT values, which weredefined as the number of cycles in the PCR reaction system whenfluorescence signals reached a set threshold. A relative expressionlevel of a target gene of the specimen was calculated byΔCT_(target)=CT_(target)−CT_(control), where CT_(target) was the CTvalue of piRNA-54265, and CT_(control) was the CT value of the formercel-miR-39 of the same specimen, so the relative expression of thetarget gene=2^(−ACTtarget).

(2) Digital PCR (based on a Bia-rad platform for example):

1) A nucleic acid content of the specimen added in 20 ul of cDNA systemwith an appropriate concentration should not exceed the detection rangespecified by digital PCR (1 to 100,000 copies of fragmented nucleic acidor 1 to 20,000 copies of complete genome DNA), and was generally 50 fgto 100 ng.

2) Configurations of the digital PCR reaction system were shown in Table16 below.

TABLE 16 Constituent Volume (μl) ddPCRProbe Supermix (no dUTP) 10piRNA-54265 forward primer 1 piRNA-54265 reverse primer 1 cel-miR-39forward primer 1 cel-miR-39 reverse primer 1 piRNA-54265 probe 1cel-miR-39probe 1 cDNA with appropriate concentration 1 Water 3 Total 20

3) After the above system configurations was completed, a digital PCRdroplet generator was enabled, a film-sealing machine was preheated, anda water-in-oil droplet generating cartridge were prepared; then 20 μl ofthe above-mentioned system in Table 16 and 70 μl of droplet-forming oilwere respectively added to avoid the generation of bubbles.

4) After a droplet-generating sealing pad was added, the mixture was putinto the digital PCR droplet generator to start generating droplets.

5) After the water-in-oil droplets were completely generated, 40 μl ofthe water-in-oil droplets were removed to a PCR reaction plate.

6) After the droplets were completely removed, the droplets were sealedwith an aluminum film. After the film was sealed, an amplificationreaction was carried out according to Table 17:

TABLE 17 Step Temperature Time Cycle Enzyme activating 95° C.  5 minutes1 Denaturizing 94° C. 30 seconds 40 Extending 60° C.  1 minute Enzymedeactivating 98° C. 10 minutes 1 Cooling 12° C. Remained —

7) The whole PCR process took about 1 hour and 45 minutes. A dropletreader was enabled to rinse and set the parameters. After theamplification was completed, the PCR reaction plate was placed on thedroplet reader to read the results.

Embodiment 13 Kit Assembly

1. A piRNA-54265 detection kit used for early screening, diagnosis,efficacy monitoring and/or prognostic evaluation of colorectal cancerincluding a piRNA-54265 detection primer pair.

The primer pair includes a forward primer and a reverse primer. Theforward primer is designed based on the sequence of the piRNA-54265(which changes U in the RNA sequence of the piRNA-54265 to T), andsequentially includes an amount of bases from the 5′⁻ of the sequence,and shall include the first five bases “TGGAG” at least. The primer pairis a piRNA-54265 stem-loop PCR primer pair, or a piRNA-54265 PolyAtailed PCR primer pair.

Preferably, the piRNA-54265 stem-loop PCR primer pair is shown as SEQ IDNO.8/9, SEQ ID NO.10/9, SEQ ID NO.11/9, SEQ ID NO.12/13, SEQ IDNO.14/13, SEQ ID NO.15/13, SEQ ID NO.16/13, SEQ ID NO.17/13, SEQ IDNO.18/13, SEQ ID NO.19/13 or SEQ ID NO.20/13.

Preferably, the piRNA-54265 PolyA tailed PCR primer pair is shown as SEQID NO.8/21, SEQ ID NO.10/21, SEQ ID NO.11/22, SEQ ID NO.12/22, SEQ IDNO.14/23, SEQ ID NO.15/22, SEQ ID NO.16/22, SEQ ID NO.17/22, SEQ IDNO.18/22, SEQ ID NO.19/22 or SEQ ID NO.20/22.

Most preferably, the forward primer and the reverse primer of thepiRNA-54265 stem-loop PCR primer pair are respectively shown as SEQ IDNO.26 and SEQ ID NO.9.

Most preferably, the forward primer and the reverse primer of thepiRNA-54265 PolyA tailed PCR primer pair are respectively shown as SEQID NO.26 and SEQ ID NO.22.

The probe sequence is show as SEQ ID NO.27.

The reverse transcription primers are classified into two types: apiRNA-54265 stem-loop reverse transcription primer or a piRNA-54265PolyA tailed reverse transcription primer.

A piRNA-54265 stem-loop reverse transcription primer sequence is shownas SEQ ID NO.2.

A piRNA-54265 PolyA tailed reverse transcription primer sequence isshown as SEQ ID NO.3-5.

2. The method of using the kit is as described in the embodiment 12.

Embodiment 14 Specimen Detection Via Kit

1. Source of specimen:

After obtaining the informed consent of the patients, serum specimens ofcolorectal cancer patients diagnosed pathologically from 2016 to 2018 inthe Sun Yat-Sen University Cancer Center and serum specimens withoutmalignant tumors found in the Cancer Prevention Physical ExaminationCenter were collected.

2. Serum piRNA-54265 levels of normal people, colorectal cancer patientsbefore onset, people with high-grade intraepithelial neoplasia incolorectum, and colorectal cancer patients were detected.

(1) According to the method in the Embodiment 12, RNAs of the serumspecimens were extracted, and then subjected to stem-loop reversetranscription, and fluorescence quantitative PCR detection.

(2) The results were shown in FIGS. 15a ˜15 b. FIG. 15a showedamplification curves of serum piRNA-54265 and external referencecel-miR-39 of one non-tumor control (CONTROL) during the follow-upvisit, one CRCcase (CASE) (no cancer at the time of sampling), one caseof high-grade intraepithelial neoplasia in colorectum, and two cases ofcolorectal cancer patients (stage I and IV).

ACt_(CRC1 Average)=6.09,ΔCt_(CRC2 Average)=10.19,ΔCt_(HIN Average)=13.84,ΔCt_(CASE Average)=15.17,and ΔCt_(CONTROL Average)=19.88.

The larger the ΔCt, the lower the relative expression amount was. Itcould be seen that the serum piRNA-54265 level of the colorectal cancerpatients was significantly higher than that of the normal people and thehigh-grade intraepithelial neoplasia patient. In addition, the serumpiRNA-54265 level of patients with colorectal cancer before diagnosiswas also higher than that of the control that no colorectal cancer wasdetected in later period.

3. The serum piRNA-54265 levels of the colorectal cancer patients beforeoperation, and in the first day, third day, fifth day, and seventh dayafter the operation were detected.

(1) According to the method in the Embodiment 12, RNAs of the serumspecimens were extracted, and then subjected to stem-loop reversetranscription, and fluorescence quantitative PCR detection.

(2) The results were shown in FIG. 16, which showed the serumpiRNA-54265 levels of the colorectal cancer patients before operation,and in the first day, the third day, the fifth day and the seventh dayafter operation. FIG. 16a showed amplification curves of the serumpiRNA-54265 and external reference cel-miR-39 of the patients beforeoperation, and in the first day, the third day, the fifth day and theseventh day after operation as well as external reference cel-miR-39.ΔCt_(before operation)=6.57,ΔCt_(in the first day after operation)=7.31,ΔCt_(in the third day after operation)=8.49,ΔCt_(in the fifth day after operation)=10.67,ΔCt_(in the seventh day after operation)=12.12. The smaller the ΔCt, thelarger the relative expression amount was. It could be seen that theserum piRNA-54265 levels decreased after operation, and the piRNA-54265levels also gradually decreased with the increase of days afteroperation.

4. Quantitative detection was performed on the serum piRNA-54265 levelsbased on a Bio-rad digital PCR platform.

(1) According to the method in the Embodiment 12, RNAs of the serumspecimens were extracted, and then subjected to stem-loop reversetranscription, and digital PCR detection.

(2) The results were as shown in FIG. 17, wherein the expression of thepiRNA-54265 could be detected both in the serums of the normal peopleand the colorectal cancer patients. After correction by reference genes,the expression levels of the piRNA-54265 in the serums of the colorectalcancer patients were significantly higher than that of the control set.

Embodiment 15 Efficacy Evaluation of piRNA-54265 Detection Kit

The experiment was designed to detect a specific amount of exogenoussynthesized piRNA-54265 analog added into a negative serum, perform RNAextraction, reverse transcription and quantitative PCR detection by thekit, investigate whether detection values of the kit accord with thereal values (a specific amount exogenously added) as well asamplification efficiency and specificity of each primer designed under aprimer design model provided by the kit.

Specific experimental method: an exogenously synthesized piRNA-54265mimic (mimic analog) was completely identical in sequence and structureto piRNA-54265 in a natural serum. A specific total amount ofpiRNA-54265 mimic (5 fmol) was added into 100 μl of negative serum(fetal calf serum), and then subjected to subsequent extraction anddetection according to the kit (external reference was required). Theresults were calculated according to the recovery calibration on the Ctvalues and the external reference.

The results were shown in FIGS. 18a-18d . FIG. 18a and FIG. 18b showedstandard curves of cel-miR-39 and piRNA-54265 standards with differentconcentrations. FIG. 18c showed the piRNA-54265 level corrected aftercalculating the recovery. The recovery was calculated by reference tothe Ct value of the cel-miR-39 detected by kit extraction of fetalbovine serum specimens added with a certain amount of cel-miR-39 and theCt value obtained by reverse transcription PCR to the cel-miR-39standards of the same amount without extraction. FIG. 18d was toevaluate the detection efficiencies of different primer pairs in FIG. 18c.

Upon the detections conducted by the kit, as shown in FIG. 18b , eachdetection result was close to the true value level, and all thedetection efficiencies were above 90%, as shown in FIG. 18d . Meanwhile,the primer pairs designed on the basis of the primer design modelprovided by the kit had both excellent amplification efficiency andspecificity (F1/R1 to F7/R7).

What is claimed is:
 1. A marker piRNA-54265 capable of being used fordiagnosing and/or treating colorectal cancer, wherein the markerpiRNA-54265 is selected from any of molecules as follows:(1) SEQ ID NO. 29: tggaggtgatgaactgtctgagcctgacc; (2) SEQ ID NO. 30:UGGAGGUGAUGAACUGUCUGAGCCUGACC; (3) SEQ ID NO. 31:GGUCAGGCUCAGACAGUUCAUCACCUCCA;

(4) a piR-54265 variant and a piR-54265 derivative modified from themolecule shown in (1), (2) or (3) with a same function; (5) a piR-54265polynucleotide construct capable of down-regulating an amount of thepiR-54265 in vivo after being introduced; and (6) an expression vectorcontaining the polynucleotide construct of (5).
 2. The markerpiRNA-54265 according to claim 1, wherein the marker piRNA-54265 is usedas a diagnostic marker, an early screening marker or a therapeutictarget for colorectal cancer.
 3. The marker piRNA-54265 according toclaim 1, wherein the marker piRNA-54265 is used as a chemosensitivityevaluation marker or a prognostic evaluation marker for a colorectalcancer patient.
 4. An expression inhibitor or a knockout reagent of themarker piRNA-54265 according to claim 1, wherein the expressioninhibitor or the knockout reagent is used as a colorectal cancertherapeutic drug or as an ancillary drug for improving thechemotherapeutic effect of colorectal cancer.
 5. The expressioninhibitor or the knockout reagent according to claim 4, wherein thecolorectal cancer therapeutic drug or the ancillary drug is a drugcapable of inhibiting proliferation, invasion and/or metastasis ofcolorectal cancer cells.
 6. A method for diagnosing/screening colorectalcancer, evaluating chemosensitivity of a colorectal cancer patient orevaluating a prognosis of a colorectal cancer patient, wherein themethod comprises detecting an expression level of piRNA-54265 in aspecimen of a subject under test, judging whether the subject suffersfrom the colorectal cancer or has a risk of suffering from thecolorectal cancer according to the expression level of the piRNA-54265,and evaluating the chemosensitivity or the prognosis of the colorectalcancer patient.
 7. A method for treating colorectal cancer, wherein themethod comprises performing gene therapy with piRNA-54265 as a target toinhibit or silence an expression of the piRNA-54265, so as to realize apurpose of treating or assisting in treating colorectal cancer.
 8. ApiRNA-54265 detection primers combination for early screening,diagnosis, curative efficacy monitoring and/or prognosis evaluation ofcolorectal cancer, wherein the piRNA-54265 detection primers combinationcomprises primer pairs for specifically detecting piRNA-54265, each ofthe primer pairs comprises a forward primer and a reverse primer, andthe forward primer is designed based on a sequence of the piRNA-54265,wherein U in the RNA sequence of the piRNA-54265 is changed to T, andsequentially comprising an amount of bases from a 5′-of the sequence,and shall comprising first five bases “TGGAG” at least.
 9. ThepiRNA-54265 detection primer combination according to claim 8, whereinthe primer pairs is a piRNA-54265 stem-loop PCR primer pair, or apiRNA-54265 PolyA tailed PCR primer pair.
 10. The piRNA-54265 detectionprimer combination according to claim 9, wherein the piRNA-54265stem-loop PCR primer pair is shown as SEQ ID NO.8/9, SEQ ID NO.10/9, SEQID NO.11/9, SEQ ID NO.12/13, SEQ ID NO.14/13, SEQ ID NO.15/13, SEQ IDNO.16/13, SEQ ID NO.17/13, SEQ ID NO.18/13, SEQ ID NO.19/13 or SEQ IDNO.20/13; and the piRNA-54265 PolyA tailed PCR primer pair is shown asSEQ ID NO.8/21, SEQ ID NO.10/21, SEQ ID NO.11/22, SEQ ID NO.12/22, SEQID NO.14/23, SEQ ID NO.15/22, SEQ ID NO.16/22, SEQ ID NO.17/22, SEQ IDNO.18/22, SEQ ID NO.19/22 or SEQ ID NO.20/22.
 11. The piRNA-54265detection primer combination according to claim 9, wherein the forwardprimer and the reverse primer of the piRNA-54265 stem-loop PCR primerpair are respectively shown as SEQ ID NO.26 and SEQ ID NO.9; and theforward primer and the reverse primer of the piRNA-54265 PolyA tailedPCR primer pair are respectively shown as SEQ ID NO.26 and SEQ ID NO.22.12. The piRNA-54265 detection primer combination according to claim 8,further comprising a probe used cooperatively with the primer pairs. 13.The piRNA-54265 detection primer combination according to claim 12,wherein the probe has a sequence shown as SEQ ID NO.27.
 14. ThepiRNA-54265 detection primer combination according to claim 8, furthercomprising a matched RNA reverse transcription primer for specimen to bedetected.
 15. A piRNA-54265 detection kit used for early screening,diagnosis, curative efficacy monitoring and/or prognostic evaluation ofcolorectal cancer, comprising the piRNA-54265 detection primercombination according to claim
 8. 16. A piRNA-54265 detection kit usedfor early screening, diagnosis, curative efficacy monitoring and/orprognostic evaluation of colorectal cancer, comprising the piRNA-54265detection primer combination according to claim
 9. 17. A piRNA-54265detection kit used for early screening, diagnosis, curative efficacymonitoring and/or prognostic evaluation of colorectal cancer, comprisingthe piRNA-54265 detection primer combination according to claim
 10. 18.A piRNA-54265 detection kit used for early screening, diagnosis,curative efficacy monitoring and/or prognostic evaluation of colorectalcancer, comprising the piRNA-54265 detection primer combinationaccording to claim
 11. 19. A piRNA-54265 detection kit used for earlyscreening, diagnosis, curative efficacy monitoring and/or prognosticevaluation of colorectal cancer, comprising the probe according to claim12.
 20. A piRNA-54265 detection kit used for early screening, diagnosis,curative efficacy monitoring and/or prognostic evaluation of colorectalcancer, comprising the probe according to claim 13.